Development of Chitosan-Coated PLGA-Based Nanoparticles for Improved Oral Olaparib Delivery: In Vitro Characterization, and In Vivo Pharmacokinetic Studies

Round 1

Reviewer 1 Report

Improving the bioavailability of drugs is a very important research topic, which is of great significance to reduce the toxic and side effects caused by high drug dosage. Recently, biodegradable polymer based nanoparticles (NPs) have gained great attention as a novel drug delivery system. In this article, authors have synthesized OLP-loaded PLGA NPs and OLP-loaded CS-PLGA NPs, and indicated that OLP-loaded PLGA NPs and OLP-loaded CS-PLGA NPs showed sustained release pattern in comparison to pure OLP suspension. In particular, CS-PLGA NPs have shown higher bioavailability and better sustained effect of the OLP. This is a useful result. In order to provide readers and colleagues with a high-quality overview, I think the article should be improved before it published in processes.

 

I think the English of the whole article should be improved, and the expression should be clearer, such as the line 211, “The FTIR spectra of OLP, PLGA, CS, OLP-loaded PLGA NPs and OLP-loaded CS-210 PLGA NPs are presented in Figure” should be “…Figure 3”.

 

In addition, many aspects should be improved, such as

1. In line 56, there should be cited more references.
2. In line 89, Table 3 seems wrong, where is the Table 1 and table 2? Line 193, table 1? Generally, they are numbered in the order in which they appear.
3. The Figures should be improved, such as Figure 6, the text on the vertical axis overlaps.
4. Uniform the format of references.

Author Response

Reviewer 1

Dear Sir/madam,

            We appreciate the reviewer for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comments and tried our best to address every one of them.

            We hope the manuscript after careful revisions meet your high standards. The authors welcome further constructive comments if any. Below we provide the point-by-point responses. All modifications in the manuscript have been done in track change.

 Query 1: I think the English of the whole article should be improved, and the expression should be clearer, such as the line 211, “The FTIR spectra of OLP, PLGA, CS, OLP-loaded PLGA NPs and OLP-loaded CS-210 PLGA NPs are presented in Figure” should be “…Figure 3”.

Answer: As suggested, As suggested, English of the manuscript has been improved. The line 210-211 has been corrected.

Query 2:  In line 56, there should be cited more references.

Answer: As suggested, a suitable references has been added in line 56.

Query 3:  In line 89, Table 3 seems wrong, where is the Table 1 and table 2? Line 193, table 1? Generally, they are numbered in the order in which they appear.

Answer: Table order in the revised manuscript has been corrected.

Query 4: The Figures should be improved, such as Figure 6, the text on the vertical axis overlaps.

Answer: Figure 6 has been improved in revised manuscript.

Query 5: Uniform the format of references.

Answer: Done

Reviewer 2 Report

In the paper 'Development of Chitosan coated PLGA-based nanoparticles for
Improved Oral Olaparib delivery: In vitro characterization, and in vivo pharmacokinetic studies ' by Md. Khalid Anwer et al., a novel approach for the preparation of Olaparib loaded NPs is presented. An extra shell of chitosan is added in order to increase the drug bioavailability.

The manuscript needs further improvements due to the following points:

• The size distribution analysis presents two peaks in the right graph, could you comment on that?
• Could you comment on the correlation between the size distibution analysis and the SEM images presented in Figure 4? 
• In FTIR spectra of Figure 2, no chitosan peak  is presented. The authors claims a peak at 282 degrees for chitosan but the graph shows data up to 240 degrees.
• The peak of OLP (FTIR spectra Figure 2) seems to be present also in the two formulations, but in the manuscript is stated the opposite; could you clarify this point?
• The drug release profile of Figure 6 seems to show no difference between the three formulations. In addition, drug release profiles seems to show lower performance compared to what reported by Misra and co-workers (doi: 10.34172/bi.2021.27).
• Please note several typos throughout the manuscript (example line 53 'systmens')

Author Response

Reviewer 2

Dear Sir/madam,

            We appreciate the reviewer for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comments and tried our best to address every one of them.

            We hope the manuscript after careful revisions meet your high standards. The authors welcome further constructive comments if any. Below we provide the point-by-point responses. All modifications in the manuscript have been done in track change.

• The size distribution analysis presents two peaks in the right graph, could you comment on that?

Answer: I have found two peaks in DLS measurement, one peak shows 97% size distribution intensity for 622 ± 9.5 nm and 2nd peak for 3% for around 250 nm. It is acceptable. Result quality showed "Good". The Z average is 622 ± 9.5 nm.

 

• Could you comment on the correlation between the size distribution analysis and the SEM images presented in Figure 4? 

Answer: The SEM images of OLP-loaded PLGA NPs and OLP-loaded CS-PLGA NPs showed homogenous population of smooth and spherical particles, with difference in size due surface coating of chitosan. The measured size nanoparticles by SEM technique were similar to those obtained by DLS.

• In FTIR spectra of Figure 2, no chitosan peak is presented. The authors claims a peak at 282 degrees for chitosan but the graph shows data up to 240 degrees.

Answer: In DSC spectra of Figure 2, chitosan peak has not be included in the spectra, the scale of chitosan peak was till 350 degree and peak appeared at 282 degree, comparative DSC spectra was taken in 50-250 degree range. So we did not included chitosan peak in this comparative scale.

• The peak of OLP (FTIR spectra Figure 2) seems to be present also in the two formulations, but in the manuscript is stated the opposite; could you clarify this point?

Answer: The FTIR spectra of OLP-loaded PLGA NPs and OLP-loaded CS-PLGA NPs did not show any prominent peaks of drug, however, some characteristics bands PLGA and chitosan could be seen, respectively. That clearly indicate encapsulation of OLP in nanocarrier.

• The drug release profile of Figure 6 seems to show no difference between the three formulations. In addition, drug release profiles seems to show lower performance compared to what reported by Misra and co-workers (doi: 10.34172/bi.2021.27).

Answer: A comparative release profile of pure OLP, OLP-PLGA NPs and OLP-CS-PLGA NPs were shown Figure 5. Free OLP drug was released quickly and achieved complete release (within 24 hours) in dissolution media. After 72 hours of exposure to the dissolution media, showed slight differences in release behavior between OLP-loaded CS-PLGA NPs and OLP-loaded PLGA NPs. OLP-CS-PLGA NPs formulation exhibited less release compared to due to coating of chitosan over PLGA nanoparticles. We are agree that our release performance shows lower performance (till 72 hours) as compared to Misra et al. (168 hours). But our release profile was well correlated with in-vivo pharmacokinetic studies.

• Please note several typos throughout the manuscript (example line 53 'systmens')

Answer: Corrected.

Reviewer 3 Report

Comments to processes-1741812

 

Line 54: Biodegradable NPs may not only increase the dissolution rate, but also the permeability of drugs across lipid base biological membrane with consequent improvement in solubility, bioavailability of drugs and reduce non-selective drug toxicity [10-12].

Line 60: But the PLGA based NPs imparts negative surface potential that destroy mucoadhesive property and decreases bioavailability of the drugs.

Why did you choose the PLGA based NPs as drug carrier? Please clarify!

 

Line 80: Briefly, OLP (30 mg) was solubilized in DMSO then added in previously prepared PLGA (100 mg) polymeric solution in dichloromethane (4 mL) in order to get oil phase.

The content of DMSO residue?

 

Line 86: Similar procedure was followed for the preparation of OLP loaded CS coated PLGA NPs, except an addition of chitosan in aqueous phase containing PVA (0.5%, w/v).

Could the CS be dissolved in aqueous solution or acid solution?

 

2.12. Statistical Analysis

and p < 0.05 was considered significant

However, there was no data compared in this MS.

Author Response

Reviewer 3

Dear Sir/madam,

            We appreciate the reviewer for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comments and tried our best to address every one of them.

            We hope the manuscript after careful revisions meet your high standards. The authors welcome further constructive comments if any. Below we provide the point-by-point responses. All modifications in the manuscript have been done in track change.

Query 1: Line 54: Biodegradable NPs may not only increase the dissolution rate, but also the permeability of drugs across lipid base biological membrane with consequent improvement in solubility, bioavailability of drugs and reduce non-selective drug toxicity [10-12].

Answer: This sentence has been rephrased.

Query 2: Line 60: But the PLGA based NPs imparts negative surface potential that destroy mucoadhesive property and decreases bioavailability of the drugs. Why did you choose the PLGA based NPs as drug carrier? Please clarify!

Answer: PLGA is the most successful and most characterized polymer for controlled release drug delivery systems. It is favored because of its biocompatibility, biodegradability and mechanical strength and continues to be used to develop new controlled release systems, and most importantly, is a FDA approved polymer

Query 3: Line 80: Briefly, OLP (30 mg) was solubilized in DMSO then added in previously prepared PLGA (100 mg) polymeric solution in dichloromethane (4 mL) in order to get oil phase. The content of DMSO residue?

Answer: The minimum volume of DMSO (0.2 mL) was used to solubilize drug that was completely removed by evaporation and also by freeze drying process.

Query 4: Line 86: Similar procedure was followed for the preparation of OLP loaded CS coated PLGA NPs, except an addition of chitosan in aqueous phase containing PVA (0.5%, w/v).

Could the CS be dissolved in aqueous solution or acid solution?

Answer: Similar procedure was followed for the preparation of OLP loaded CS coated PLGA NPs, except chitosan was dissolved in aqueous phase containing PVA (0.5%, w/v) in 1% v/v acetic acid.

Query 5:  2.12. Statistical Analysis

and p < 0.05 was considered significant

However, there was no data compared in this MS.

Answer: The statistical analysis was performed in pharmacokinetic parameters comparison as mentioned in section 3.7 and data presented in Table 3.

Reviewer 4 Report

1. The abstract must be rewritten, it's not understood easily.
2. The introduction part needs some clarifications on why Chitosan coated PLGA-based nanoparticles for

Improved Oral Olaparib delivery? what are the advantages of this work compared to prior works?

 

3. The drug release studies of prepared NPs were performed in phosphate buffer (pH- 7.4) only. Also, it should study in pH-5, 6.8, and 8 . because in a cancerous tumor, normal cells, blood serum all pH conditions are different.

 

4. For morphological study TEM images are also needed along with SEM images, It will improve your manuscript quality.

 

5. "3.1. Measurement of particle size, PDI and zeta potential" here author mentioned particle sizes but it should be hydrodynamic particle size.

 

6. "3.5. Morphology of nanoparticles 223

"The OLP loaded PLGA-NPs appeared smooth, spherical particles with a narrow size of distribution On the other hand, OLP loaded CS-PLGA-NPs appeared spherical and smooth surface particles but bigger in size, probably due to coating of chitosan (Figure 4).". How authors discussed without any proof about narrow sizes. Here need a particle distributions graphs.

 

7. Throughout the manuscript, English should be rewritten with proper sentences. Many grammatical mistakes and sentences are not made properly. like " A sharp endothermic peak was exhibited by pure OLP at 217.56 °C [28], confirming crystalline for of drug..

Also, throughout the manuscript, especially in the "3. Results and Discussion" sections. The authors misuse scientific terms. Lack of Scientific discussions with proper evidence.

Author Response

Reviewer 4

Dear Sir/madam,

            We appreciate the reviewer for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comments and tried our best to address every one of them.

            We hope the manuscript after careful revisions meet your high standards. The authors welcome further constructive comments if any. Below we provide the point-by-point responses. All modifications in the manuscript have been done in track change.

Query 1:  The abstract must be rewritten, it's not understood easily.

Answer: As suggested, abstract has been improved.

Query 2:  The introduction part needs some clarifications on why Chitosan coated PLGA-based nanoparticles for Improved Oral Olaparib delivery? what are the advantages of this work compared to prior works?

Answer:  Due mucoadhesive characteristic of CS, it facilitates the interaction with negatively charged membranes and mucosa, that promoting adhesion and retention of the drug. In addition, CS has ability to open the tight junction of the intestinal epithelium and promoting the permeability.  The bioavailability of OLP was enhanced by 2 and 4.75 times in OLP-loaded PLGA NPs and OLP-loaded CS-PLGA NPs, respectively that was significantly higher compared to previously reported work by Akshay et.al, who developed nanoliposhphere and nanosuspension of OLP.

Query 3:  The drug release studies of prepared NPs were performed in phosphate buffer (pH- 7.4) only. Also, it should study in pH-5, 6.8, and 8, because in a cancerous tumor, normal cells, blood serum all pH conditions are different.

Answer: We selected the pH of drug release media (pH-7.4) that is favorable for tumor targeting. pH of cancer cells is arounfd 7.4  as reported by Arora et al., 2012.

[Arora, S.; Saharan, R.; Kaur, H.; Kaur, I.; Bubber, P.; Bharadwaj, L.M. Attachment of Docetaxel to Multiwalled Carbon Nanotubes for Drug Delivery Applications. Adv.Sci.Lett. 2012, 17, 70-75. https://doi.org/10.1166/asl.2012.4251

Query 4:  For morphological study TEM images are also needed along with SEM images, It will improve your manuscript quality.

Answer: We appreciate the suggestion of the reviewer. Right now, we do not have TEM access in our university. In future studies of nanoparticle, we will include TEM images along with SEM images.

Query 5: "3.1. Measurement of particle size, PDI and zeta potential" here author mentioned particle sizes but it should be hydrodynamic particle size.

 Answer: Thanks for this comments, It has been corrected in revised manuscript.

 Query 6: "3.5. Morphology of nanoparticles 223

"The OLP loaded PLGA-NPs appeared smooth, spherical particles with a narrow size of distribution On the other hand, OLP loaded CS-PLGA-NPs appeared spherical and smooth surface particles but bigger in size, probably due to coating of chitosan (Figure 4).". How authors discussed without any proof about narrow sizes. Here need a particle distributions graphs.

Answer: The section 3.5 morphology of nanoparticles has been improved in revised manuscript. The PDI values of OLP loaded PLGA-NPs and OLP loaded CS-PLGA-NPs were measured as 0.360 and 0.321 (Table 2) by DLS method respectively, which confirmed narrow size distribution.

Query 7: Throughout the manuscript, English should be rewritten with proper sentences. Many grammatical mistakes and sentences are not made properly. like " A sharp endothermic peak was exhibited by pure OLP at 217.56 °C [28], confirming crystalline for of drug.

Answer: As suggested, English of manuscript has been improved throughout the manuscript.

Query 8:  Also, throughout the manuscript, especially in the "3. Results and Discussion" sections. The authors misuse scientific terms. Lack of Scientific discussions with proper evidence.

Answer: As suggested, Results and Discussion section has been improved with proper citation.

 

Round 2

Reviewer 2 Report

Dear authors,

Thanks for providing an answer to my questions/doubts. I would like to follow up with additional comments.

The fact that the second peak is at 250 nm is due to the addition of chitosan, could you please explain why the addition of chitosan induce the appearance of a second peak? if not, could you elaborate a possible explanation?

As far as I can see, both SEM images are showing a quite broad particle distribution. The most interesting aspect is that the max of distribution seems to be close to 1 um in diameter. This is not correlating with your finding in the DLS analysis and the dimension is far from being 'nano'.

In the DSC spectra of OLP loaded CS-PLGA-NPs is the peak of chitosan still present at 282 degrees? REF 19 does not show any reference spectra of chitosan so for sake of clarity you should include an appropriate reference or your reference spectrum in the SI.

From the FTIR spectra is difficult to conclude that there is a clear encapsulation of the OLP in a CS-PLGA-NPs structure. Unless more studies are performed or more reference literature provided, there is no evidence of the encapsulation of OLP. The final sentence 'That clearly indicate encapsulation of OLP in nanocarrier.' is just speculative and it is not supported by clear evidences.

As stated in the mauscript introduction, the goal of this approach is to increase the bioavailability of the drug within the first 24 hours after subministration. you confimed that during the first 24 hours, there is no difference in the drug release profile so I am wondering what is the information and the novelty Figure 5 is bringing to the manuscript.

Author Response

Dear Sir/madam,

            We appreciate the reviewer for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comments and tried our best to address every one of them.

            We hope the manuscript after careful revisions meet your high standards. The authors welcome further constructive comments if any. Below we provide the point-by-point responses. All modifications in the manuscript have been done in track change with yellow highlights.

 

Thanks for providing an answer to my questions/doubts. I would like to follow up with additional comments.

The fact that the second peak is at 250 nm is due to the addition of chitosan, could you please explain why the addition of chitosan induce the appearance of a second peak? if not, could you elaborate a possible explanation?

Answer: The possible explanation can be the existence of a single-sized OLP loaded CS-PLGA-NPs with the 250 nm, the 622 nm representing the nanoparticle agglomeration. The other possibility could be the coexistence of two types of nanoparticles with different sizes.

As far as I can see, both SEM images are showing a quite broad particle distribution. The most interesting aspect is that the max of distribution seems to be close to 1 um in diameter. This is not correlating with your finding in the DLS analysis and the dimension is far from being 'nano'.

Answer: We respectfully disagree with this comments. SEM images of OLP loaded PLGA-NPs and OLP loaded CS-PLGA-NPs clearly showing the approximate size of 392 nm and 622 nm respectively, that correlates with DLS results.

In the DSC spectra of OLP loaded CS-PLGA-NPs is the peak of chitosan still present at 282 degrees? REF 19 does not show any reference spectra of chitosan so for sake of clarity you should include an appropriate reference or your reference spectrum in the SI.

Answer: Thank you for the input. It was a mistake. The reference [19] has been replaced with a suitable reference in revised manuscript.

From the FTIR spectra is difficult to conclude that there is a clear encapsulation of the OLP in a CS-PLGA-NPs structure. Unless more studies are performed or more reference literature provided, there is no evidence of the encapsulation of OLP. The final sentence 'That clearly indicate encapsulation of OLP in nanocarrier.' is just speculative and it is not supported by clear evidences.

Answer: As suggested, suitable references has been added in revised manuscript.

As stated in the mauscript introduction, the goal of this approach is to increase the bioavailability of the drug within the first 24 hours after subministration. you confimed that during the first 24 hours, there is no difference in the drug release profile so I am wondering what is the information and the novelty Figure 5 is bringing to the manuscript.

Answer: Free OLP drug was released within 24 hours. The release was sustained for 72 hours in both OLP loaded PLGA-NPs and OLP loaded CS-PLGA-NPs to enhance the efficacy of drug. However, due to charge attraction between OLP and chitosan in OLP loaded CS-PLGA-NPs exhibited more sustained release of drug in comparison to OLP loaded PLGA-NPs

Reviewer 3 Report

OK

Author Response

Dear Sir/madam,

            We appreciate the your precious time in reviewing. Thanks for acceptance.

Regard

Dr. Anwer

Reviewer 4 Report

Query 3: The drug release studies of prepared NPs were performed in phosphate buffer (pH- 7.4) only. Also, it should study in pH-5, 6.8, and 8, because in a cancerous tumor, normal cells, blood serum all pH conditions are different.

Answer: We selected the pH of drug release media (pH-7.4) that is favorable for tumor targeting. pH of cancer cells is arounfd 7.4 as reported by Arora et al., 2012.

 

Please see the Abstract of the same reference (arora et. aL 2012). ALL OVER THE MANUSCRIPT they never mentioned cancerous cells pH 7.4.  Please again read this published article and must follow it. Otherwise, it will great mistake for science and misrepresentation. 

Abstract: In this manuscript we report the development of multiwalled carbon nanotubes—docetaxel conjugate by covalent interaction, involving nucleophilic substitution reaction mechanism. The surface functionalization of nanotubes was carried out by enrichment of carboxylic groups with optimized oxidation treatment. The carboxyl groups were converted to acyl groups followed by covalent attachment of docetaxel to multiwalled carbon nanotubes with a cleavable ester linkage. The drug carbon nanotube conjugate was characterized by Fourier Transform Infra red, UV-Visible, Raman spectroscopy, Scanning and Transmission electron microscopy. The drug release from the conjugate was studied in vitro in phosphate buffers having pH 5, 6.8 and 7.4. It was demonstrated that conjugate showed drug release faster in acidic pH, which resembles the pH of cancerous cells, as compared to buffer of normal cell pH. This property would reduce the drug dosage thereby substantially benefit the antitumor effect and would boost significantly the applications of carbon nanotubes in the biomedicine field.

Query 2:  The introduction part needs some clarifications on why Chitosan coated PLGA-based nanoparticles for Improved Oral Olaparib delivery? what are the advantages of this work compared to prior works?

Answer:  Due mucoadhesive characteristic of CS, it facilitates the interaction with negatively charged membranes and mucosa, that promoting adhesion and retention of the drug. In addition, CS has ability to open the tight junction of the intestinal epithelium and promoting the permeability.  The bioavailability of OLP was enhanced by 2 and 4.75 times in OLP-loaded PLGA NPs and OLP-loaded CS-PLGA NPs, respectively that was significantly higher compared to previously reported work by Akshay et.al, who developed nanoliposhphere and nanosuspension of OLP.

"Akshay et.al" is only one comparative prior work. Need some more References. 

Query 5: "3.1. Measurement of particle size, PDI and zeta potential" here author mentioned particle sizes but it should be hydrodynamic particle size.

 Answer: Thanks for this comments, It has been corrected in revised manuscript.

Still in Table-2, and Figure 1, mentioned particle size, Which should be "Hydrodynamic particle size". Hydrodynamic particle size is always greater than the real sizes of particles. 

 

 

Author Response

Dear Sir/madam,

            We appreciate the reviewer for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comments and tried our best to address every one of them.

            We hope the manuscript after careful revisions meet your high standards. The authors welcome further constructive comments if any. Below we provide the point-by-point responses. All modifications in the manuscript have been done in track change with yellow highlights.

 

Query 3: The drug release studies of prepared NPs were performed in phosphate buffer (pH- 7.4) only. Also, it should study in pH-5, 6.8, and 8, because in a cancerous tumor, normal cells, blood serum all pH conditions are different.

Answer: We selected the pH of drug release media (pH-7.4) that is favorable for tumor targeting. pH of cancer cells is around 7.4 as reported by Arora et al., 2012.

 Please see the Abstract of the same reference (arora et. aL 2012). ALL OVER THE MANUSCRIPT they never mentioned cancerous cells pH 7.4.  Please again read this published article and must follow it. Otherwise, it will great mistake for science and misrepresentation. 

Abstract: In this manuscript we report the development of multiwalled carbon nanotubes—docetaxel conjugate by covalent interaction, involving nucleophilic substitution reaction mechanism. The surface functionalization of nanotubes was carried out by enrichment of carboxylic groups with optimized oxidation treatment. The carboxyl groups were converted to acyl groups followed by covalent attachment of docetaxel to multiwalled carbon nanotubes with a cleavable ester linkage. The drug carbon nanotube conjugate was characterized by Fourier Transform Infra red, UV-Visible, Raman spectroscopy, Scanning and Transmission electron microscopy. The drug release from the conjugate was studied in vitro in phosphate buffers having pH 5, 6.8 and 7.4. It was demonstrated that conjugate showed drug release faster in acidic pH, which resembles the pH of cancerous cells, as compared to buffer of normal cell pH. This property would reduce the drug dosage thereby substantially benefit the antitumor effect and would boost significantly the applications of carbon nanotubes in the biomedicine field.

Answer: Thanks for pointing this comments out, Authors agree that pH 7.4 and pH 6.8 is pH of normal and cancerous cells, respectively, reported by Arora et al. We did not quoted this references in our manuscript.

Query 4:  The introduction part needs some clarifications on why Chitosan coated PLGA-based nanoparticles for Improved Oral Olaparib delivery? what are the advantages of this work compared to prior works?

Answer:  Due mucoadhesive characteristic of CS, it facilitates the interaction with negatively charged membranes and mucosa, that promoting adhesion and retention of the drug. In addition, CS has ability to open the tight junction of the intestinal epithelium and promoting the permeability.  The bioavailability of OLP was enhanced by 2 and 4.75 times in OLP-loaded PLGA NPs and OLP-loaded CS-PLGA NPs, respectively that was significantly higher compared to previously reported work by Akshay et.al, who developed nanoliposhphere and nanosuspension of OLP..

"Akshay et.al" is only one comparative prior work. Need some more References. 

Answer: As suggested, one more reference has been added in revised manuscript.

Query 5: "3.1. Measurement of particle size, PDI and zeta potential" here author mentioned particle sizes but it should be hydrodynamic particle size.

 Answer: Thanks for this comments, It has been corrected in revised manuscript.

Still in Table-2, and Figure 1, mentioned particle size, Which should be "Hydrodynamic particle size". Hydrodynamic particle size is always greater than the real sizes of particles. 

Answer: Corrected.

 

 

Round 3

Reviewer 2 Report

It would be nice if some of the hypothesis presented by the authors to the reviewers will be included in the text together with appropriate references.

Author Response

Dear Sir/madam,

            We appreciate the reviewer for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comments and tried our best to address every one of them.

 

It would be nice if some of the hypothesis presented by the authors to the reviewers will be included in the text together with appropriate references.

Answer: Already added with suitable references.

Reviewer 4 Report

Answer: Thanks for pointing this comments out, Authors agree that pH 7.4 and pH 6.8 is pH of normal and cancerous cells, respectively, reported by Arora et al. We did not quoted this references in our manuscript. 

Why not quoted/ Author should keep this reference. 

Author Response

Dear Sir/madam,

            We appreciate the reviewer for your precious time in reviewing our paper and providing valuable comments. It was your valuable and insightful comments that led to possible improvements in the current version. The authors have carefully considered the comment and tried our best to address every one of them.

 

Why not quoted/ Author should keep this reference. 

Answer: As suggested, Arora et.al, reference has been added in the text and list of references.

Round 4

Reviewer 4 Report

The release profile of pure OLP, OLP-PLGA NPs and OLP-CS-PLGA NPs must be tested at pH 5, 6.4, and 7.4. Which I mentioned Last 3 times. Otherwise manuscript will not be scientific properly.    

Author Response

Query: The release profile of pure OLP, OLP-PLGA NPs and OLP-CS-PLGA NPs must be tested at pH 5, 6.4, and 7.4. Which I mentioned Last 3 times. Otherwise manuscript will not be scientific properly.

Answer: It was reported that the conjugate showed faster drug release in acidic region (pH 5.0), likely to the condition of cancerous cells, than in normal cell condition (pH 7.4) [25]. In this study, the experiments were done at pH 7.4, observation at acidic condition would be needed in further study.